The present invention relates generally to genes expressing selectable phenotypes. More particularly, the present invention relates to genes capable of co-expressing both dominant positive selectable and negative selectable phenotypes.
Genes which express a selectable phenotype are widely used in recombinant DNA technology as a means for identifying and isolating host cells into which the gene has been introduced. Typically, the gene expressing the selectable phenotype is introduced into the host cell as part of a recombinant expression vector. Positive selectable genes provide a means to identify and/or isolate cells that have retained introduced genes in a stable form, and, in this capacity, have greatly facilitated gene transfer and the analysis of gene function. Negative selectable genes, on the other hand, provide a means for eliminating cells that retain the introduced gene.
A variety of genes are available which confer selectable phenotypes on animal cells. The bacterial neomycin phosphotransferase (neo) (Colbere-Garapin et al., J. Mol. Biol. 150:1, 1981), hygromycin phosphotransferase (hph) (Santerre et al., Gene 30:147, 1984), and xanthine-guanine phosphoribosyl transferase (gpt) (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981) genes are widely used dominant positive selectable genes. The Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977); the cellular adenine phosphoribosyltransferase (APRT) (Wigler et al., Proc. Natl. Acad. Sci. USA 76:1373, 1979); and hypoxanthine phosphoribosyltransferase (HPRT) genes (Jolly et al., Proc. Natl. Acad. Sci. USA 80:477, 1983) are commonly used recessive positive selectable genes. In general, dominant selectable genes are more versatile than recessive genes, because the use of recessive genes is limited to mutant cells deficient in the selectable function, whereas dominant genes may be used in wild-type cells.
Several genes confer negative as well as positive selectable phenotypes, including the HSV-I TK, HPRT, APRT and gpt genes. These genes encode enzymes which catalyze the conversion of nucleoside or purine analogs to cytotoxic intermediates. The nucleoside analog ganciclovir (GCV) is an efficient substrate for HSV-I TK, but a poor substrate for cellular TK, and therefore may be used for negative selection against the HSV-I TK gene in wild-type cells (St. Clair et al., Antimicrob. Agents Chemother. 31:844, 1987). However, the HSV-I TK gene may only be used effectively for positive selection in mutant cells lacking cellular TK activity. Use of the HPRT and APRT genes for either positive or negative selection is similarly limited to HPRTxe2x88x92 or APRTxe2x88x92 cells, respectively (Fenwick, xe2x80x9cThe HGPRT Systemxe2x80x9d, pp. 333-373, M. Gottesman (ed.), Molecular Cell Genetics, John Wiley and Sons, New York, 1985; Taylor et al., xe2x80x9cThe APRT Systemxe2x80x9d, pp. 311-332, M. Gottesman (ed.), Molecular Cell Genetics, John Wiley and Sons, New York, 1985). The gpt gene, on the other hand, may be used for both positive and negative selection in wild-type cells. Negative selection against the gpt gene in wild-type cells is possible using 6-thioxanthine, which is efficiently converted to a cytotoxic nucleotide analog by the bacterial gpt enzyme, but not by the cellular HPRT enzyme (Besnard et al., Mol. Cell. Biol. 7:4139, 1987).
Another negatively selectable gene has recently been reported by Mullen et al., Proc. Natl. Acad. Sci. USA 89:33, 1992. The bacterial cytosine deaminase (CD) gene converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). 5-FU is further metabolized intracellularly to 5-fluoro-uridine-5xe2x80x2-triphosphate and 5-fluoro-2xe2x80x2-deoxy-uridine-5xe2x80x2-monophosphate, which inhibit RNA and DNA synthesis, causing cell death. Thus, 5-FC can effectively ablate cells carrying and expressing the CD gene. The CD gene is not positively selectable in normal cells.
More recently, attention has turned to selectable genes that may be incorporated into gene transfer vectors designed for use in human gene therapy. Gene therapy can be used as a means for augmenting normal gene function, for example, by introducing a heterologous gene capable of modifying cellular activities or cellular phenotype, or alternatively, expressing a drug needed to treat a disease. Gene therapy also is a method for permanently curing a hereditary genetic disease which results from a defect in or absence of one or more genes. Collectively, such diseases result in significant morbidity and mortality. Examples of such genetic diseases include hemophilias A and B (caused by a deficiency of blood coagulation factors VIII and IX, respectively), alpha-1-antitrypsin deficiency, and adenosine deaminase deficiency. In each of these particular cases, the missing gene has been identified and its complementary DNA (cDNA) molecularly cloned (Wood et al., Nature 312:330, 1984; Anson et al., Nature 315:683, 1984; and Long et al., Biochemistry 23:4828, 1984; Daddona et al., J. Biol. Chem. 259:12101, 1984). While palliative therapy is available for some of these genetic diseases, often in the form of administration of blood products or blood transfusions, one way of permanently curing such genetic diseases is to introduce a replacement for the defective or missing gene back into the somatic cells of the patient, a process referred to as xe2x80x9cgene therapyxe2x80x9d (Anderson, Science 226:401, 1984).
The process of gene therapy typically involves the steps of (1) removing somatic (non-germ) cells from the patient, (2) introducing into the cells ex vivo a therapeutic or replacement gene via an appropriate vector capable of expressing the therapeutic or replacement gene, and (3) transplanting or transfusing these cells back into the patient, where the therapeutic or replacement gene is expressed to provide some therapeutic benefit. Gene transfer into somatic cells for human gene therapy is presently achieved ex vivo (Kasid et al., Proc. Natl. Acad. Sci. USA 87:473, 1990; Rosenberg et al., N. Engl. J. Med. 323:570, 1990), and this relatively inefficient process would be facilitated by the use of a dominant positive selectable gene for identifying and isolating those cells into which the replacement gene has been introduced before they are returned to the patient. The neo gene, for example, has been used to identify genetically modified cells used in human gene therapy.
In some instances, however, it is possible that the introduction of genetically modified cells may actually compromise the health of the patient. The ability to selectively eliminate genetically modified cells in vivo would provide an additional margin of safety for patients undergoing gene therapy, by permitting reversal of the procedure. This might be accomplished by incorporating into the vector a negative selectable (or xe2x80x9csuicidexe2x80x9d) gene that is capable of functioning in wild-type cells. Incorporation of a gene capable of conferring both dominant positive and negative selectable phenotypes would ensure co-expression and co-regulation of the positive and negative selectable phenotypes, and would minimize the size of the vector. However, positive selection for the gpt gene in some instances requires precise selection conditions which may be difficult to determine. Moreover, the feasibility of using the gpt gene for in vivo negative selection has not yet been clearly established. For these reasons, co-expression of a dominant positive selectable phenotype and a negative selectable phenotype is typically achieved by co-expressing two different genes which separately encode other dominant positive and negative selectable functions, rather than using the gpt gene.
The existing strategies for co-expressing dominant positive and negative selectable phenotypes encoded by different genes often present complex challenges. As indicated above, the most widely used technique is to co-transfect two plasmids separately encoding two phenotypes (Wigler et al., Cell 16:777, 1979). However, the efficiency of co-transfer is rarely 100%, and the two genes may be subject to independent genetic or epigenetic regulation. A second strategy is to link the two genes on a single plasmid, or to place two independent transcription units into a viral vector. This method also suffers from the disadvantage that the genes may be independently regulated. In retroviral vectors, suppression of one or the other independent transcription unit may occur (Emerman and Temin, Mol.Cell. Biol. 6:792, 1986). In addition, in some circumstances there may be insufficient space to accommodate two functional transcription units within a viral vector, although retroviral vectors with functional multiple promoters have been successfully made (Overell et al., Mol. Cell. Biol. 8:1803, 1988). A third strategy is to express the two genes as a bicistronic mRNA using a single promoter. With this method, however, the distal open reading frame is often translated with variable (and usually reduced) efficiency (Kaufman et al., EMBO J. 6:187, 1987), and it is unclear how effective such an expression strategy would be in primary cells.
The present invention provides a method for more efficiently and reliably co-expressing a dominant positive selectable phenotype and a negative selectable phenotype encoded by different genes.
The present invention provides a selectable fusion gene comprising a dominant positive selectable gene fused to and in reading frame with a negative selectable gene. The selectable fusion gene encodes a single bifunctional fusion protein which is capable of conferring a dominant positive selectable phenotype and a negative selectable phenotype on a cellular host. In a preferred embodiment, the selectable fusion gene comprises nucleotide sequences from the hph gene fused to nucleotide sequences from the HSV-I TK gene, referred to herein as the HyTK selectable fusion gene (SEQ ID NO:1. The HyTK selectable fusion gene confers both hygromycin B resistance (Hmr) for dominant positive selection and ganciclovir sensitivity (GCVs) for negative selection.
In another preferred embodiment, the selectable fusion gene comprises nucleotide sequences from the bacterial CD gene fused to nucleotide sequences from the neo gene, referred to herein as the CD-neo selectable fusion gene (SEQ ID NO:3. The CD-neo selectable fusion gene confers both G-418 resistance (G-418r) for dominant positive selection and 5-fluorocytosine sensitivity (5-FCs) for negative selection.
The present invention also provides recombinant expression vectors, for example, retroviruses, which include the selectable fusion genes, and cells transduced with the recombinant expression vectors.
The selectable fusion genes of the present invention are expressed and regulated as a single genetic entity, permitting co-regulation and co-expression with a high degree of efficiency.